Lactobacillus acidophilus is a Gram-positive, rod-shaped, non-spore forming, homofermentative bacterium that is a normal inhabitant of the gastrointestinal and genitourinary tracts. Since its' original isolation by Moro (1900) from infant feces, the “acid loving” organism has been found in the intestinal tract of humans, breast-fed infants, and persons consuming high milk, lactose, or dextrin diets. Historically, L. acidophilus is the Lactobacillus species most often implicated as an intestinal probiotic capable of eliciting beneficial effects on the microflora of the gastrointestinal tract (Klaenhammer and Russell (2000) “Species of the Lactobacillus acidophilus complex,” Encyclopedia of Food Microbiology, Volume 2, pp. 1151-1157. Robinson et al. eds. (Academic Press, San Diego, Calif.). L. acidophilus can ferment hexoses, including lactose and more complex oligosaccharides, to produce lactic acid and lower the pH of the environment where the organism is cultured. Acidified environments (e.g., food, vagina, and regions within the gastrointestinal tract) can interfere with the growth of undesirable bacteria, pathogens, and yeasts. The organism is well known for its acid tolerance, survival in cultured dairy products, and viability during passage through the stomach and gastrointestinal tract. Lactobacilli and other commensal bacteria, some of which are considered as probiotic bacteria that favor life, have been studied extensively for their effects on human health, particularly in the prevention or treatment of enteric infections, diarrheal disease, prevention of cancer, and stimulation of the immune system. Genetic characterization of other Lactobacillus species (e.g., L johnsonii and L. rhamnosus) has been described (see e.g., U.S. Pat. No. 6,476,209; U.S. Pat. No. 6,544,772; U.S. Patent Publication Nos. 20020159976, 2003013882 & 20040009490; PCT Publication No. WO 2004/031389; PCT Publication No. 2003/084989; PCT Publication No. WO 2004/020467).
Lactic acid bacteria are widely used for the production of fermented milk products. Their requirement for an exogenous source of amino acids or peptides necessitates having an efficient proteolytic system that can degrade the casein in milk into the necessary small peptides and single amino acids used for growth. The peptides and amino acids generated through proteolysis are also involved in the development of texture and flavor in dairy products. Enzymes of the proteolytic system include a cell wall-bound extracellular proteinase (CEP), which is responsible for the initial breakdown of casein, and various intracellular peptidases, which further degrade the oligopeptides thus formed. In addition, there are proteins involved in amino acid transport systems for the uptake of peptides and amino acids from the environment, and enzymes involved in converting amino acids into flavor compounds.
There are two main types of CEPs, designated PI and PIII (Visser et al. (1986) Appl. Environ. Microbiol. 52:1162; Siezen (1999) Antonie Van Leeuwenhoek 76:139-55). The multi-domain, cell-envelope proteinases encoded by the genes prtB of Lactobacillus delbrueckii subsp. bulgaricus, prtH of Lactobacillus helveticus, prtP of Lactococcus lactis, scpA of Streptococcus pyogenes and csp of Streptococcus agalactiae have been compared using multiple sequence alignment, secondary structure prediction and database homology searching methods. This comparative analysis has led to the prediction of a number of different domains in these cell-envelope proteinases, and their homology, characteristics and putative function are described. These domains include, starting from the N-terminus, a pre-pro-domain for secretion and activation, a serine protease domain (with a smaller inserted domain), two large middle domains A and B of unknown but possibly regulatory function, a helical spacer domain, a hydrophilic cell-wall spacer or attachment domain, and a cell-wall anchor domain. Not all domains are present in each cell-envelope proteinase, suggesting that these multi-domain proteins are the result of gene shuffling and domain swapping during evolution.
The CEPs differ in their cleavage specificity toward caseins, with PI preferentially degrading β-casein, not α- or κ-casein, and PIII degrading α-, β-, and κ-caseins (Pritchard and Coolbear (1993) FEMS Micro. Rev. 12:179-206). Less bitterness was generated from casein degraded by a PIII-type proteinase than by a PI-type proteinase (Visser et al. (1983) Neth. Milk Dairy J. 17:169-175). The domains mapped from various lactic acid bacteria CEPs include the pre-pro-domain for secretion and activation, a serine protease or catalytic domain, two large middle domains A and B which are thought to have a regulatory and stabilizing function, a helical spacer domain, a hydrophilic cell wall spacer domain, and a cell wall anchor domain (Siezen (1999) Antonie Leeuwenhoek 76:139-155). The cell wall anchor contains an LPXTG (SEQ ID NO:135) sequence that is cleaved after translocation, and the enzyme is thought to be covalently linked to the peptidoglycan layer. This anchor is not present in the CEPs of some lactobacillus species.
Peptidases include aminopeptidases, dipeptidases, proline-specific peptidases, tripeptidases, carboxypeptidases, and endopeptidases. The peptidases have overlapping substrate specificities, and three or more peptidases need to be disrupted simultaneously to observe an effect on growth rate in milk (Mierau et al. (1996) J. Bacteriol. 179:2794-2803). Aminopeptidases are capable of releasing single amino acid residues from oligopeptides, and are therefore important for flavor development in fermented milk products (Law and Haandrikman (1997) Int. Dairy J. 7:1-11). In cheese production, for example, it is thought that lysis of the starter bacteria releases peptidases into the curd, which then hydrolyze the casein-derived peptides into amino acids, resulting in enhanced flavor (Meijer et al. (1998) Appl. Env. Micro. 64:1950-1953).
Because of the roles peptidases play, peptidase gene sequences are needed for genetic modification of bacteria, particularly Lactobacillus. 